Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo.

Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor in clinical trials as a cancer therapy that has been recently shown to block human immunodeficiency virus Tat transactivation and viral replication through inhibition of positive transcription elongation factor b (P-TEFb). Flavopiridol is the most potent P-TEFb inhibitor reported and the first Cdk inhibitor that is not competitive with ATP. We examined the ability of flavopiridol to inhibit P-TEFb (Cdk9/cyclin T1) phosphorylation of both RNA polymerase II and the large subunit of the 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and found that the IC(50) determined was directly related to the concentration of the enzyme. We concluded that the flavonoid associates with P-TEFb with 1:1 stoichiometry even at concentrations of enzyme in the low nanomolar range. These results indicate that the apparent lack of competition with ATP could be caused by a very tight binding of the drug. We developed a novel immobilized P-TEFb assay and demonstrated that the drug remains bound for minutes even in the presence of high salt. Flavopiridol remained bound in the presence of a 1000-fold excess of the commonly used inhibitor DRB, suggesting that the immobilized P-TEFb could be used in a simple screening assay that would allow the discovery or characterization of compounds with binding properties similar to flavopiridol. Finally, we compared the ability of flavopiridol and DRB to inhibit transcription in vivo using nuclear run-on assays and concluded that P-TEFb is required for transcription of most RNA polymerase II molecules in vivo.

Flavopiridol inhibits P-TEFb and blocks HIV-1 replication.

Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor that is in clinical trials as a cancer treatment because of its antiproliferative properties. We found that the flavonoid potently inhibited transcription by RNA polymerase II in vitro by blocking the transition into productive elongation, a step controlled by P-TEFb. The ability of P-TEFb to phosphorylate the carboxyl-terminal domain of the large subunit of RNA polymerase II was inhibited by flavopiridol with a K(i) of 3 nm. Interestingly, the drug was not competitive with ATP. P-TEFb composed of Cdk9 and cyclin T1 is a required cellular cofactor for the human immunodeficiency virus (HIV-1) transactivator, Tat. Consistent with its ability to inhibit P-TEFb, flavopiridol blocked Tat transactivation of the viral promoter in vitro. Furthermore, flavopiridol blocked HIV-1 replication in both single-round and viral spread assays with an IC(50) of less than 10 nm.

Surgical management of refractory exit-site/tunnel infection of Tenckhoff catheter: technical innovations of partial replantation.

OBJECTIVE: Catheter-related infection has been the major cause of catheter removal for peritoneal dialysis (PD) patients. A salvage technique--partial replantation of the infected catheter--was developed in our hospital to rescue catheters with refractory exit-site or tunnel infection. PATIENTS: We performed 26 partial replantations of Tenckhoff catheters for 23 patients with refractory exit-site or tunnel infection and 2 patients with near-cuff perforation of the catheter. Their problems were all resolved successfully without interruption of PD. INTERVENTIONS: We removed the infected portion of the catheter and preserved the still-functioning internal conduit, connecting it to a divided new catheter. All of the patients resumed PD immediately after the advancement of the new catheter through a new subcutaneous tunnel and exit site on the opposite side. RESULTS: No technical complications such as disconnection of the catheter or leakage of dialysate were noted. Repeated partial replantation of the catheter was done for 1 patient with a new refractory exit-site infection. Tunnel infection was not an absolute contraindication for this procedure. About one third (34.6%) of our patients had preoperative tunnel infection. CONCLUSION: Partial replantation of a Tenckhoff catheter is a simple and effective procedure for patients with refractory exit-site/tunnel infection and patients with near-cuff perforation of the catheter. Repeated partial replantation is also feasible for repeat exit-site infections.

Transmural suture technique for trocar-site bleeding following laparoscopic cholecystectomy.

Laparoscopic cholecystectomy was performed in a cirrhotic patient who had cholelithiasis. Despite the existence of coagulopathy, excessive bleeding from the gallbladder and nodular liver was avoidable. Dissection and extraction of the gallbladder went smoothly. However, serious bleeding from the trocar site occurred following the withdrawal of the trocar/cannula. The bleeding was not controllable by electrocauterization. A novel attempt using a transmural suture technique was tried, and hemostasis was achieved satisfactorily. Our patient enjoyed an uneventful postoperative recovery and was discharged 2 days after surgery.

In vivo antithrombotic effect of triflavin, an Arg-Gly-Asp containing peptide on platelet plug formation in mesenteric microvessels of mice.

Triflavin, an Arg-Gly-Asp-containing snake venom peptide, inhibits platelet aggregation through the blockade of fibrinogen binding to the activated platelets. In this study, platelet thrombus formation was induced by irradiation of the mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. Electron microscopy reveals moderately damaged endothelial cells, as well as aggregates consisting almost exclusively of platelets with pseudopod formation, and degranulated appearance. Triflavin (10-20 micrograms/g) significantly prolonged the lag period of inducing platelet plug formation in mesenteric venules when it was intravenously infused. Triflavin (20 micrograms/g) prolonged the occlusion time about 2-fold (from control 112 +/- 23 to 240 +/- 47 s). Furthermore, PGE1 briefly prolonged the occlusion time about 1.5-fold (from 105 +/- 21 to 168 +/- 20 s) when it was given by continuous infusion (40 micrograms/kg/min). On the other hand, triflavin was also effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at dose of 2-4 micrograms/g. Heparin (1.5 U/g) and indomethacin (200 micrograms/g) had no significant effect in prolonging the occlusion time or in reducing ADP-induced pulmonary embolism in mice. Therefore, triflavin is an effective antithrombotic agent in preventing the thromboembolism in these two in vivo models.

Embolization of a renal transplant pseudoaneurysm following angiolipoma resection. A case report.

The development of a pseudoaneurysm in renal allografts is a well-known complication of percutaneous biopsy. However, the authors report a case of pseudoaneurysm formation in a renal cadaver allograft, following documented angiolipoma resection prior to transplantation. Treatment required superselective embolization with multiple platinum coils.

Laparoscopic assisted gastrostomy tube placement.

Gastrostomy commonly is performed for patients who have esophageal stenosis or difficulty in swallowing. The fact that most patients who require a gastrostomy are malnourished or debilitated urges surgeons to seek a safe and simple procedure. Percutaneous endoscopic gastrostomy (PEG) was introduced into clinical use and recommended as the procedure of choice in the 1980s. However, for patients with obstruction of the oroesophageal canal, PEG was not possible, and only traditional open gastrostomy could be considered. We describe a technique for placement of a gastrostomy tube under laparoscopic guidance. The technique is simple and easily mastered, requires equipment routinely used in laparoscopic surgery, and can be used by surgeons without gastroendoscopic experience. When performed properly, the method is simple, economical, and reliable.

Laparoscopic-guided gastrostomy.

Gastrostomy is commonly performed in patients who have esophageal stenosis or difficulty in swallowing. The fact that most patients who require a gastrostoma are usually malnourished or debilitated urges surgeons to seek a convenient and reliable method for performing the operation. Percutaneous endoscopic gastrostomy (PEG) was introduced into clinical practice and recommended as the procedure of choice in the 1980s. However, PEG was not indicated and only traditional open gastrostomy was considered for those who had obstruction in their oroesophageal canal. We describe a technique for placement of a gastrostomy tube under laparoscopic control. The technique is easily mastered, requires equipment routinely used in laparoscopic control. The technique is easily mastered, requires equipment routinely used in laparoscopic surgery, and can be used by surgeons untrained in gastroendoscopy. When performed properly, the method is accurate, economic and reliable.

Revision of dysfunctional Tenckhoff catheters by laparoscopic technique in continuous ambulatory peritoneal dialysis patients.

A laparoscopic technique was used to rescue dysfunctional Tenckhoff catheters in five continuous ambulatory peritoneal dialysis (CAPD) patients. This method proved to be effective in recovering the function of the catheters, whose malfunction was found to be due to catheter entrapment or endoluminal tamponing by the omentum. The videoscope-assisted laparoscopic technique provides access and allows identification and eradication of the specific etiology leading to catheter drainage failure. It also provides an alternative for treating patients with a dysfunctional Tenckhoff catheter. This method avoids the need to remove the original catheter or to reimplant a new catheter.

A technique using V-shaped transmural suture fixation in percutaneous endoscopic gastrostomy.

A new method of percutaneous endoscopic gastrostomy (PEG) using V-shaped transmural suture fixation was performed on 2 patients with advanced carcinoma. This method secures the gastrostoma in a way superior to the conventional technique. It facilitates exchange of the catheter and avoids potential morbidity caused by peritoneal leakage even when tube feeding has been initiated in the early post-insertion stage. Inadvertent complications of hematoma formation and necrotizing fasciitis can also be prevented.

Activation of troponin C by Cd2+ and Pb2+.

Certain heavy metal actions such as Cd2+ and Pb2+ mimic Ca2+ effectively in stimulating calmodulin (CaM). We now show that these cations also activate skeletal muscle troponin C (TnC), a Ca2(+)-binding protein highly homologous to CaM. Like Ca2+, these cations allow TnC to alter its electrophoretic mobility on polyacrylamide gels, and to bind to phenyl-Sepharose. Moreover, they activate TnC to stimulate myofibrillar ATPase. When TnC was removed from the skeletal myofibrils by treatment with trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CDTA), the ATPase activity was no longer stimulated by the cations. However, after reconstitution of CDTA-treated skeletal myofibril with TnC, the response of ATPase to Ca2+, Cd2+ or Pb2+ was restored. These findings suggest that the activation of myofibrillar ATPase by Cd2+ and Pb2+ is mediated through TnC. The ability of the heavy metals to stimulate TnC-supported ATPase activity correlated quite well with the ability to increase the extent of the myofibrillar superprecipitation. The activation of TnC by Cd2+ or Pb2+ could constitute a possible molecular basis for their toxicity.

Preoperative evaluation of rectal carcinoma invasion using transrectal ultrasonography: preliminary experience of 30 patients.

Transrectal ultrasonography was used to examine and to preoperatively evaluate rectal carcinoma invasion in 30 patients. Satisfactory sonography pictures were obtained in 23 patients. Four stages of tumor invasion were identified in our study. The sensitivity of preoperative evaluation in this study was 91%. In order to verify the correspondence between the ultrasonographic findings and the actual histo-anatomic condition of rectum, 10 resected rectal specimens were studied by in vitro ultrasonography. We conclude that the appearance of the muscularis propria layer of rectum as a homogeneous hypoechogenic band can be stably defined with transrectal ultrasonography and is crucial for accurate evaluation of cancer invasion. Although lymph node enlargement was shown as hypoechogenic nodules on the sonographic pictures, the existence of cancer metastasis could not be differentiated in this study.

Stimulation of calmodulin by cadmium ion.

Cd2+, a serious environmental pollutant in certain industrial regions, accumulates in mammalian tissues with a very slow turnover. Using various criteria, we studied the ability of Cd2+ to substitute for Ca2+ in calmodulin (CaM), a ubiquitous Ca2+-binding protein that mediates many of the Ca2+ effects. CaM bound Cd2+ with a Kd of 4.5 microM, presumably to the Ca2+-binding sites. Binding of Cd2+ allowed CaM to bind 2 moles chlorpromazine, or to form a complex with skeletal muscle troponin-I, troponin-T, or phosphodiesterase. Complex formation with phosphodiesterase led to its activation, which was observed even in the presence of glutathione or cysteine, agents known to chelate Cd2+. This raises the possibility that one manifestation of Cd2+ toxicity may be through its activation of CaM, thus upsetting its normal regulation by a cellular flux of Ca2+.

Fecal Salmonella carriers in the Chinese army recruits in Taiwan.

A survey for Salmonella carriers in the Chinese army recruits was made. Each isolated strain was identified by conventional biochemical tests, rapid Inolex plates, and for identification serological methods. The rapid Inolex plates could save approximately 24-48 hours for identification. Salmonella was detected in 1.83% of the rectal swabs collected from 1,150 recruits and 50 cooks. Among the 22 isolates 5, 1, 5, and 6 strains were identified as Salmonella groups: B, C1, D and E1 respectively and 5 strains were ungroupable. Salmonella isolates were found to be susceptible to chloramphenicol, kanamycin, ampicillin and tetracycline. Chloramphenicol or kanamycin is still the drug of choice for treatment of Salmonella infections, but the former should be used with caution.